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1. Expression of circadian clock gene Period 2 in the mammalian bladder identified by real-time imaging with luciferase reporter gene Changhao Wu*, Guiping Sui, Simon Archer, Ying Chen, University of Surrey, UK 259 Introduction Background:The circadian masterclock including the Period 2 (Per2) gene in central nervous system regulates sleep cycle and other rhythmic activities. Expression of Per2 has also been found in peripheral tissues. Diurnal urine production has been widely described and most recently nocturnal variations in urodynamics have been reported. However, little is known about the circadian clock in the bladder. Objective: To explore whether Per2 gene is expressed in the mammalian bladder. Methods The transgenic knock-in Per2::Luciferase mice on a C57Bl/6J background was used to report the expression of Per2. The mice contain a fusion protein of Per2 and Luciferase. When Per2 is translated in the presence of the substrate luciferin, its expression can be monitored as bioluminescence. The cyclic variation in Per2 expression from mouse bladders incubated in culture medium was continuously monitored in real time for 24hrs. Prior to the experiments, mice were maintained at a 12:12 hour light to dark cycle. • Conclusions • This study demonstrates for the first time that mammalian bladders • express Per2 proteins. • Per2 expression exhibits circadian rhythm. • It also responds to mechanical and chemical stimuli. • These intrinsic Per2 activities may have a role in regulating bladder • function. A dysregulated Per2 expression could potentially underlie • bladder abnormalities such as nocturia and detrusor over-activity. Figure 1. Expression of Per2 in the mouse urinary bladders. Per2 expression was measured by luminescence emission of the reporter gene luciferase and expressed as relative light unit (RLU). Mean±SEM from 7 knock-in Per2::Luciferase mice bladders vs wild-type control (n=4). Figure 2. Variations of the Per2 expression in a 24 hour cycle. Mean ± SEM of Per2:: luciferase activity from 5 bladders; Note: peak at 12 th hour. Figure 3. Peak and trough values of the Per2 expression in a 24 hour cycle. B A Figure 4. Effect of mechanical stimulus on Per2:luciferase activity. Mechanical disturbance by turning the sample vial upside down three times increased the light intensity. Figure 5. Effect of chemical stimuli on Per2 expression. A. Cholinergic (carbachol) and purinergic (ATP-γ-S) neurotransmitters; B. Intracellular Ca2+ chelator –BAPTA-AM. Note: a shift of the circadian peak by the neurotransmitters. n=4.